Holography was invented over 60 years ago by the physicist Dennis Gabor and is a technique that allows the light scattered from an object to be recorded and later reconstructed. Digital holography uses digital reconstruction of the diffraction patterns.
In digital holographic microscopy, a diffraction pattern, obtained by interference between a reference wave and an object wave which has interacted with an object of interest, may be detected and stored in a digital recording. By applying a reconstruction algorithm to such a recorded diffraction pattern, an image or image signature of the object of interest may be obtained. Coherent or partially coherent light, collimated by a small aperture, may be used to illuminate an object in order to produce a diffraction pattern. This diffraction pattern may then be acquired by a high resolution optoelectronic sensor array. Such a lens-free holographic microscopy setup may produce a hologram of the object with phase information encoded in the diffraction images. Lens-free holographic imaging can provide an attractive low-cost solution for imaging small objects, for example, microscopic objects, such as biological cells, because no expensive or complex optical components, such as high-quality optical lenses, are required.
Methods for holographic imaging in biological applications known in the art may be primarily based on in-line transmission geometry, in which coherent light from a light source illuminates a sample, which may be positioned on a glass substrate, and the diffraction or fringe pattern is recorded on an imager which is positioned on the opposite side of the sample with respect to the light source.
FIG. 1 illustrates an exemplary holography setup, which may be known in the art, for creating a holographic image of a transparent object. This setup comprises a light source 102, an aperture 105 (e.g., a pin-hole), a transparent surface 106 (e.g., a glass substrate) for supporting an object 104, and an image sensor 101. The aperture 105 may collimate a light wave 107 emanating from the light source 102 to produce a substantially planar parallel coherent or partially coherent light wave near the object 104 after propagating unimpeded over a suitable distance between the aperture 105 and the object 104. The light wave may then interact with the object 104, for example, the object may undergo a phase shift due to changes in refractive index while passing through the object. The diffraction pattern formed by interference of an object wave component, which has interacted with the object 104, and a reference wave component, which has passed through the transparent surface 106 without interacting with the object 104, may then be recorded by the image sensor 101.
In a paper by Su et al., published in Lab Chip, 2009, 9, 777-787, a lens-free holographic cytometer is disclosed. This paper describes an imaging and reconstruction method that may result in an improvement of the reconstructed images, by providing rich texture information. This system is furthermore used for characterization and counting of cells which are positioned on a CMOS imaging chip. The paper therefore demonstrates that identification and/or characterization of a heterogeneous cell solution on a chip is feasible based on pattern recognition of the holographic diffraction pattern of each cell type.
However, holographic imaging using an in-line transmission geometry may not be suitable for imaging non-transparent samples. Furthermore, dense or connected objects, such as biological tissue samples, may prevent the undistorted transmission of a suitable fraction of the wave through the sample in order to form a reference wave component. Therefore, when imaging such a non-transparent or dense sample, a suitable object wave component may preferentially be obtained by reflection on the surface of the sample, instead of transmission through the sample.
When a high resolution for small objects needs to be achieved, the reflective-mode setup may involve a complicated set-up. FIG. 2 illustrates the working principles of a field portable reflection/transmission microscope based on lens-less holography, which was disclosed in a paper by Lee et al., published in Biomedical Optics Express, 2011, 2(9), 2721-2730. The configuration of this setup is similar to that of a Michelson interferometer, and comprises a light source 102, an image sensor 101 (e.g., a CMOS sensor-chip), a reflective surface 103, and a beam splitting device 108. It demonstrates a lens-less reflection-mode microscope based on digital off-axis holography in which a beam-splitter 108 and a reflective surface 103 are used to produce a tilted reference wave for producing an interference pattern by superposition on the reflected light from the object 104. Therefore, an off-axis hologram of the object 104 is created on the image sensor 101. The beam-splitter 108 is an essential feature of the device for the interference of the reflected beam with the reflected light from an object to create the hologram.